By the early 1970's, natural killer (NK) cell activity, distinct from antigen specific cytolytic T lymphocyte activity, had been reported by several laboratories in a variety of species, including mouse, rat and man. These reports are reviewed by R. K. Oldham, J. Biol. Resp. Mod. 1,217 (1982). Non-specific cytotoxic cells (NCC), similar in many aspects to NK cells, also have been identified in lower vertebrate species (Graves et al, Dev. Comp. Immunol. 8,293 (1984); Evans et al, Dev. Comp. Immunol. 8,303 (1984); Evans et al., Dev. Comp. Immuno. 8,599 (1984) Evans et al, Dev. Comp. Immunol. 8, 823 (1984).
NK cells are known to be a heterogeneous population of immune cells with regard to phenotype and function, as discussed, for example, by Lanier et al, Immunol. Today, 7,132 (1986). However, controversy still exists with regard to their lineage with respect to other immune cells and to their relationship to other effector cells such as lymphokine-activated killer (LAK) cells and anomolous killer (AK) cells. These differences may reflect the innate heterogeneity of the types of cells capable of NK activity (as defined by function) or be due to the various assay systems employed to study NK cells.
NK cells have been implicated in a variety of activities involving the immune system, including immune disorders in both animal models and man. Results from animal models have also shown that NK cells are effective in vivo against the growth and metastasis of certain types of tumors. In this regard, administration of in vitro cultured NK cells to human cancer patients has shown promise in the treatment and regression of certain types of malignancies. Further, NK cells have been implicated in host resistance to infections with microorganisms, both bacteria and viruses. Finally, NK cells are thought to play an important role in the normal regulation of the host immune system through immunoglobulin production and hematopoiesis. These lines of evidence suggest that the NK system possesses considerable functional diversity, and may operate by the recognition of changes in normal membrane structures, including differentiation antigens, as discussed by Toshitani et al, Cell Immunol.108,188 (1987); Kornbluth et al, J. Immunol. 134,728 (1985); Lauzon and Roder, Cell Immunol. 94,85 (1985); Roder et al, J. Exp. Med. 150,471 (1979).
Although NK cells have been studied for several decades, two of the major questions that remain in NK biology today are what molecule(s) on the surface of NK cells is involved in recognition of the target cells and what molecule(s) on the target cells is recognized by NK cells. At the beginning of the last decade it was found that T cells recognize products of the major histocompatibility complex (MHC) expressed on target cells. This discovery was facilitated by the availability of monoclonal antibodies (mAbs) against these molecules which inhibited their recognition by T cells. In terms of NK biology, mAb inhibition of function or recognition (in the absence of complement) has been much less frequently documented. Both the transferring receptor and the receptor for IgG Fc have been implicated to serve as recognition structures for NK cells, although these results are controversial (Vodinelich et al, Proc. Natl. Acad. Sci. USA 80,835 (1983); Dokhelar et al, Eur. J. Immunol. 14,340 (1984). The laminin/laminin receptor complex has also recently been implicated to act as a means of NK recognition of certain cells, as reported at the Fourth Int'l Workshop on NK Cells, Kingston, Ontario, 1986. Finally, carbohydrate/carbohydrate interactions have been implicated to serve as a means of NK/target cell recognition by Muchmore et al, Immunobiol. 158,191 (1981); Werkmeister et al, Cell. Immunol. 80,172 (1983). Together, these results have been interpreted as signifying that NK cells do not express clonally-restricted receptors, that NK cells may express more than one receptor on their surface and that NK cells are capable of recognizing multiple antigens on the surface of one or more target cells. These results may also be indicative of NK heterogeneity due to discrete NK subpopulations.
The NK cell membrane antigens and mAbs capable of detecting these determinants are shown in Table 1.
TABLE 1 ______________________________________ Natural killer cell membrane antigens and mAbs capable of detecting these determinants. mAb Designation Antigen Specificity ______________________________________ CD2 (Leu-5b) T cells, NK cells CD7 (Leu-9) T cells, NK cells CD8 (Leu-2) T cells, NK cells CD11 (Leu-15) C3bi receptor on T Cells, NK cells, monocytes and macrophages CD16 (Leu-11a) Fc (gamma) receptor on NK cells and on PMN's Leu 7 HNK-1 on LGL's and NK cells Leu 19 NKH-1 determinant (220 KD) on NK and T-cells .sup.a OKT8 T cytotoxic/suppressor cells and LGL OKT10 LGL, early thymus antigen 3A1 T cells and LGL 5A12 T cells and LGL Lyt 3 T cells and LGL OKT5 T cytotoxic/suppressors and LGL Ia Activated T cells ______________________________________ .sup.a Cells expressing OKT8, OKT5 and Ia are nonlytic LGL's.
Although typical NK activity is generally accepted to be mediated by cells with the Leu4- (CD3-), Leu11+ (CD16+) and Leu19+ surface phenotype, there is compelling evidence that typical T cells, particularly antigen-specific cytotoxic T cells (CTL), can mediate NK-like cytotoxicity as measured against certain tumor cells such as K562, as described by Brooks et al., Immunol. Rev. 72, 43 (1983) and others. These CTLs are referred to as CTL(NK) or non-MHC-restricted CTL. Since it has been shown that true NK cells do not transcribe mRNA for or express on their surface a typical T cell antigen receptor (TCR) (Reynolds et al, J. Exp. Med. 150,471 (1985); Lanier et al, J. Exp. Med. 163,209 (1986); Tutt et al, J. Immunol. 137,2998 (1986)), it is generally accepted that NK cells do not recognize antigen in this fashion and do not recognize MHC molecules. Although the CTL(NK) do express a typical TCR, it is not known whether this molecule is utilized to effect their NK-like killing or if these effector cells recognize MHC antigens in some aberrant fashion. Recently, there has been evidence to suggest that the CD3 molecule and the gamma/delta TCR may be involved in this type of non-MHC-restricted cytotoxicity (Farrini et al, J. Exp. Med. 166,277 (1987); Alarcon et al, Proc. Natl. Acad. Sci. USA 84,3861 (1987); Ang et al, J. Exp. Med. 165,1453 (1987); David et al, J. Immunol. 138,2831 (1987); van de Grien et al, J. Immunol.138,1627 (1987). In other studies, it has been demonstrated that anti-CD3 and anti-CD8 mAb's, while inhibiting the antigen-specific lysis by such CTL clones, do not inhibit the NK-like lysis by these effector cells (Moretta et al, Eur. J. Immunol. 14,121 (1984); Brooks and Holscher, J.Immunol. 6,594 (1987); Roberts and Moore, Eur. J. Immunol. 15,448 (1985).
Very little is known regarding identification of lymphoreticular cells in fish. Previous studies have tentatively identified a B lymphocyte subpopulation in this species. However, there have been only a few investigations where attempts have been made to identify cytotoxic cells in teleost fish. In mammals, however, a great deal is known regarding the heterogeneity of lymhocytes and mAbs have been derived which inhibit cytotoxic activity. For example, anti-OKT3 mAbs identify a determinant found on most human T cells. Monoclonal antibodies specific for this antigen inhibit CTL killing of Epstein-Barr virus infected cells. The OKT10 determinant is present on human natural killer cells as well as on other cells and anti-OKT10 mAbs inhibit NK lysis of K652, MOLT-4, and CEM tumor targets. Monoclonal antibodies directed against the glycoprotein T-200, an antigen that is present on all human peripheral blood T-lymphocytes, blocks the ability of K562 cells to inhibit NK killing of MOLT-4 cells in cold target inhibition assays. The mAb NK-8 inhibits LGL mediated cytotoxicity against K562 cells by 50 to 60%. Two mAbs (RH 7.2 and 17.2) directed against alloimmune human cytotoxic lymphocytes inhibit NK cytolysis when added at the Ca.sup.++ pulse phase.
It therefore seems apparent that in order to understand the process of NK cell recognition, it is essential to identify and characterize examples of NK target antigens, as well as the receptor(s), in a variety of species, including lower vertebrates such as fish and mammals.
Knowledge of an NK antigen receptor would enable analysis of how the receptor/ligand complex functions. Further, isolation of an NK antigen receptor would allow comparison with other receptors and possibly the identification of other NK antigen receptors or subclasses of NK cells on the basis of antigen specificity and/or type of antigen receptor. This would further understanding of the relationship between NK, LAK and CTL(NK) populations as well as immune cells such as monocytes, macrophages and other types of T cells. A comparison of the receptor molecules in fish, mouse and humans provides insights into the evolution of the NK cell population and the role they play in immune function, malignancy and immune disorders.
Knowledge of NK target antigens allows comparisons with other known ligands recognized by effector cells, such as MHC molecules, and could lead to the identification of other target antigens that may be similar in normal host defense, tumor biology and NK cell regulation. Further, as true for the NK cell receptor, identification of an NK target antigen could serve to classify NK cells into discrete subpopulations based on "antigen specificity", in that there may be subpopulations of NK cells which preferentially recognize certain target antigens. Knowledge of these molecules may have important implications concerning the types of antigen receptors present on other types of nonspecific effector cells. An analysis of the target antigen at the biochemical level may lead to a better understanding of what types of molecules are important in NK cell recognition and the role of these cells in the immune system. A comparison of the target antigens in fish, mouse and man at the biological, biochemical and molecular levels should give some insight into the evolution of the NK population in the immune system and possibly as to what challenges to the immune system this system evolved to combat. That is, if these target antigens are very conserved, at all levels of analysis, this may indicate the existence of important regulatory structures in the host and thus strong evolutionary pressures on the NK system to maintain a method by which to recognize these molecules. The anti-target cell mAb's may be of in vivo relevance in that, if NK cells are important in defense against malignancy, then an understanding of the recognition and regulation of this antigen may be useful in the detection and treatment of cancer via screening for malignancy and immune disorders, and for targeting of NK cells or mAbs to tumors.
It is therefore an object of the present invention to provide antibodies which specifically recognize and bind to antigens on the surface of NK cells and cells lysed by NK cells.
It is a further object of the present invention to provide such antibodies which can inhibit the lysis of target cells by NK cells.
It is a still further object of the present invention to provide antibodies which are useful in the isolation and characterization of NK cell populations as defined by their antigen receptors and molecules recognized thereby, as well as the actual antigens.